The majority of drugs under development target cell surfaces. Examples include molecules that bind G-protein coupled receptors and ion channels, bivalent monoclonal antibody (mAb) or engineered multivalent mAb fragments that recognize cell surface antigens, and other small molecules or nano-particles that attack disease cells based on multivalent recognition. In view of this cell-centered drug development, there is a growing need of high throughput analytical techniques incorporating the fluidic cell membrane environment. Fluidic supported lipid bilayers (SLBs) constitute a benchmark in studying cell surface processes, providing an analytical platform which mimics the cell membrane environment [PNAS, 1984, 81, 6159-6163; Science, 1996, 271, 43-48; Acc. Chem. Res. 2002, 35, 149-157; Nature, 2005, 437, 656-663]. However, due to instability of the SLB under a variety of sample processing or handling conditions, particularly exposure to air [J. Phys. Chem. B 1999, 103, 2554-2559], developing the SLB into a practical and high-throughput technique has been extremely difficult.
Wagner and Tamm [Biophys. J. 2000, 79, 1400-1414;], Knoll et al. [Rev. Mol. Biotech. 2000, 74, 137-158], Naumann et al. [Biomacromolecules 2002, 3, 27-35], and U.S. Pat. Nos. 5,922,594, 6,756,078, and 7,045,171 teach the art of producing supported lipid bilayer membranes with improved stability by covalently tethering a fraction of lipid molecules in the membrane via spacers to polymer cushions or self-assembled monolayer coated solid substrate surfaces, but these tethered supported lipid bilayers did not show air stability. Several strategies have been attempted to provide air stability to the supported lipid membrane. These include: 1) hybrid bilayer [U.S. Pat. No. 5,919,576; Langmuir, 1999, 15, 5128-5135] or biomimetic membrane [Langmuir 1999, 15, 3866-3874] where a stable lipid monolayer assembles onto an alkanethiol self-assembled monolayer or a hydrophobic polymer brush; 2) polymerized membrane from diacetylene-lipid conjugates [Langmuir, 2003, 19, 1752-1765]; 3) protein protected membrane bilayer [J. Am. Chem. Soc. 2004, 126, 6512-6513]; 4) membrane bilayers containing poly(ethyleneglycol) (PEG) conjugated lipids [Langmuir 2005, 21, 7476-7482.]; and 5) trehalose protected membrane bilayers [J. Am. Chem. Soc. 2007, 129, 10567-10574.]. The membrane from approach 1) does not allow the incorporation of transmembrane proteins and thus has limited use. The membrane from approach 2) lacks the lipid mobility necessary for mimicking cell surface functions. The surfaces of membrane bilayers from approaches 3) & 4) are covered with a layer of protecting protein molecules or PEG brushes that may prevent proper interaction of membrane receptors with their targets. In approach 5), which is adapted from anhydrobiotic organisms [Nature Biotech. 2000, 18, 145-146], the supported lipid bilayer becomes air-stable if the solution in contact with the supported lipid bilayer before drying contains a relatively high concentration of trehalose. This approach requires the introduction of a large amount of trehalose into the solution every time the supported lipid bilayer is exposed to air and is not practical in bioanalysis.
Fang et al. teach the art of fabricating air-stable membrane protein microarrays on polar or reactive surfaces, particular γ-aminopropylsilane (GAPS) coated glass surfaces [US 2004/0096914 A1; J. Am. Chem. Soc. 2002, 124, 2394-2395], while McBee and Saavedra reported that SUVs deposited on the GAPS surface did not form SLBs and did not possess sufficient stability upon withdrawing of the sample from the air-water interface [Langmuir 2005, 21, 3396-3399]. The approach of Fang et al. was based on immobilizing protein molecules in membrane fractions. A similar approach was demonstrated by Giess et al. in the so-called “protein-tethered lipid bilayer” [Biophys. J. 2004, 87, 3213-3220]. A disadvantage of these approaches based on immobilized proteins is the lack of protein mobility within the supported membrane. Protein mobility is essential to many cell membrane processes. An excellent example is the interaction of integrin receptors with adhesion proteins at the extracellular matrix (ECM). Binding of integrin receptors to Arg-Gly-Asp (RGD) moiety on ECM proteins triggers not only conformational change in the integrin receptor, but also receptor clustering and dynamic increase in the valency of cell adhesion, a process termed “adhesion strengthening” [Curr. Opin. Cell. Biol. 2003, 15, 547-56; Science 1992, 255, 1671-1677.]. Another example is the immune system, which distinguishes “self” from “non-self” based on the dynamic and multivalent recognition by T-lymphocytes of specific antigenic peptides displayed by the major histocompatibility complex (MHC) on the surface of antigen-presenting cells [Science, 1999, 285, 221-227]. Immobilizing protein molecules may also lead to the loss of protein activity, as demonstrated by Cha et al. [Proteomics, 2005, 5, 416-419].
It is apparent that there is a need for improved methods to produce supported lipid bilayer membranes with the following properties: (i) air-stable; (ii) possessing a high level of fluidity which allows receptor or ligand clustering; (iii) free from obstructions to membrane surface interactions; and (iv) easily implemented in an array format. The present invention utilizes tethered sterols groups incorporated into the bottom leaflet of the supported lipid bilayer membrane to impart the properties listed above. Sterols, particularly cholesterol, are predominant and naturally occurring components of plasma membranes of mammalian and eukaryotes cells. The presence of cholesterol increases the stability and rigidity of lipsomes by increasing their area-expansion modulus and bending energies [Biophys. J. 2006, 90, 1639-1649]. The rigid and flat cholesterol molecule imposes conformational ordering locally and increases the packing density of lipid molecules in the immediate surrounding [Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 269-295]. In nature, cholesterol molecules in the cell membrane are known to cluster and phase separate into cholesterol rich and cholesterol deficient domains. These cholesterol clusters only stabilize surrounding lipids in the cholesterol rich domains, but not cholesterol deficient domains. To avoid phase separation in a supported lipid bilayer and to fully take advantage of the stabilizing effect, we immobilize cholesterol groups (also referred to as cholesteryl groups) on a solid support; the immobilized and dispersed cholesterol groups interact with the entire bottom leaflet of a supported lipid bilayer, thus imparting the desired property of air-stability. Air-stability was not achieved in previous studies of supported lipid bilayers on tethered cholesteryl groups in binary thiol self-assembled monolayers (SAMs) on gold surfaces [Langmuir 1998, 14, 839-844] where the mobility of the thiolate anchor and the extensive phase separation in the self-assembled monolayer resulted in domains of cholesteryl thiol separated from those of short hydrophilic thiols [Sensors & Actuators B 2007, 124, 501-509]. Vesicle deposition on such a phase separated surface led to lipid monolayers on the cholesteryl domains while lipid bilayers formed on the hydrophilic thiol domains, without achieving air-stability.